MCHR1基因shRNA真核表达载体的构建及鉴定

Construction and identification of shRNA recombinant plasmids targeting MCHR1 gene

目的构建有效针对小鼠黑色素浓集激素受体1(melanin-concentrating hormone receptor 1,MCHR1)的shRNA(small hairpin RNA)真核表达载体。方法设计合成3条小鼠MCHR1基因的shRNA序列以及1条无同源性的shRNA序列作为阴性对照,与质粒重组构建sh-MCHR1干扰载体,并进行菌液PCR和DNA测序鉴定;脂质体法转染小鼠3T3-L1细胞后观察MCHR1 mRNA和蛋白表达的变化。结果经菌液PCR及DNA测序鉴定表明各shRNA载体构建成功;转染3T3-L1细胞后,与空载体组相比,阴性对照组MCHR1 mRNA和蛋白的表达均无明显差异,但实验组3种干扰载体均能显著抑制MCHR1 mRNA和蛋白的表达(P<0.05),且3种载体的抑制程度有一定的差异,以sh-MCHR1-1载体沉默效果最佳。结论 sh-MCHR1干扰载体构建成功,为进一步探究MCHR1与肥胖症之间的关系奠定了实验基础。

Objective To construct effective small hairpin RNA （shRNA） recombinant plasmids targeting mouse melanin-concentrating hormone receptor 1 （MCHR1） gene. Methods Three shRNA sequences targeting mouse MCHR1 gene and one negative control were designed, synthesized and then recombined with plasmid. The shRNA recombinant vectors were detected by bacterium liquid PCR reaction and DNA sequencing. These vectors were also transfected into 3T3-L1 preadipocytes with lipofeetamine, and RT-PCR and Western blot analysis were performed to evaluate their silencing effects. Results Every shRNA recombinant vector was constructed and transfected into 3T3-L1 preadipoeytes successfully. Compared with empty vector group, three vectors can significantly inhibit the expression of mRNA and protein of MCHR1 （P 〈 0. 05 ）, and negative control vector had no obvious change. In addition, the effects of the three vectors were different, and the sh-MCHRI-1 produced the best. Conclusion The shRNA recombinant vectors targeting mouse MCHR1 gene were established successfully, and it will contribute to further study on the relationship between MCHR1 and obesity.